Research Article

Co-Amorphous Combination of Nateglinide-Metformin Hydrochloridefor Dissolution Enhancement

Sarika Wairkar1,2 and Ram Gaud1

Received 7 May 2015; accepted 15 July 2015; published online 28 August 2015

Abstract. The aim of the present work was to prepare a co-amorphous mixture (COAM) of Nateglinideand Metformin hydrochloride to enhance the dissolution rate of poorly soluble Nateglinide. Nateglinide(120 mg) and Metformin hydrochloride (500 mg) COAM, as a dose ratio, were prepared by ball-millingtechnique. COAMs were characterized for saturation solubility, amorphism and physicochemical interac-tions (X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), Fourier transforminfrared spectroscopy (FTIR)), SEM, in vitro dissolution, and stability studies. Solubility studies revealeda sevenfold rise in solubility of Nateglinide from 0.061 to 0.423 mg/ml in dose ratio of COAM. Solid-statecharacterization of COAM suggested amorphization of Nateglinide after 6 h of ball milling. XRPD andDSC studies confirmed amorphism in Nateglinide, whereas FTIR elucidated hydrogen interactions(proton exchange between Nateglinide and Metformin hydrochloride). Interestingly, due to low energyof fusion, Nateglinide was completely amorphized and stabilized by Metformin hydrochloride.Consequently, in vitro drug release showed significant increase in dissolution of Nateglinide in COAM,irrespective of dissolution medium. However, little change was observed in the solubility and dissolutionprofile of Metformin hydrochloride, revealing small change in its crystallinity. Stability data indicated notraces of devitrification in XRPD of stability sample of COAM, and % drug release remained unaffectedat accelerated storage conditions. Amorphism of Nateglinide, proton exchange with Metformin hydro-chloride, and stabilization of its amorphous form have been noted in ball-milled COAM of Nateglinide-Metformin hydrochloride, revealing enhanced dissolution of Nateglinide. Thus, COAM of Nateglinide-Metformin hydrochloride system is a promising approach for combination therapy in diabetic patients.

KEY WORDS: COAM; dose ratio; improved Nateglinide dissolution; Metformin hydrochloride;Nateglinide.

INTRODUCTION

Drug safety and therapeutic efficacy are critical concernsof the bioavailability of drugs. Thus, poor solubility and sub-sequently poor oral bioavailability of a drug have become amajor challenge in the formulation industry (1,2). The currentscenario reveals abundant research carried out on theamorphization of drugs, a state which possesses high thermo-dynamic energy and significantly improved Bapparent^ solu-bility. However, such a structurally disordered metastableform causes free molecular mobility and enhanced chemicalreactivity too (3). Therefore, stabilization of the amorphousform is a vital issue as the thermodynamic drive is preordainedtowards devitrification during the manufacturing process andshelf life of the product (4,5).

In order to stabilize amorphous drugs, solid polymericdispersions have been widely attempted and extensive

literature is available on the mechanism of drug-polymerdispersions, molecular interactions therein, and subsequentstabilization (6–8). In some cases, the use of polymers mayinhibit the re-crystallization of drugs by increasing glass tran-sition temperature (Tg) as compared to a pure amorphousdrug (9,10). It is also assumed that molecular interactionsbetween drug and polymer play an important role in stabili-zation of the drug. However, amorphous dispersions of drug-polymer are typically hygroscopic, and the absorbed moisturecan reduce the Tg of the dispersion followed by re-crystallization of the drug (11). Manufacturing of aforesaiddispersions is critical due to the amount of polymers involvedand limited miscibility of drugs into polymers leading to abulkier dosage form (12,13).

Binary co-amorphous mixture formation has been ex-tensively studied for a decade towards the stabilization ofamorphous drugs, which includes the use of small mole-cules like saccharine, amino acids, citric acid, and sugars(14–16). Firstly, binary amorphous mixtures of two drugswere reported by Yamamura et al. (17) for cimetidine andindomethacin, in which the amorphous precipitates wereobtained by solvent evaporation method. Recently, Radeet al. have developed co-amorphous binary mixtures of

1 Shobhaben Pratapbhai Patel School of Pharmacy & TechnologyManagement, SVKMs NMIMS, V.L.Mehta Road, Vile Parle (W),Mumbai, Maharashtra 400 056, India.

2 To whom correspondence should be addressed. (e-mail:sarikawairkar@gmail.com)

AAPS PharmSciTech, Vol. 17, No. 3, June 2016 (# 2015)DOI: 10.1208/s12249-015-0371-4

673 1530-9932/16/0300-0673/0 # 2015 American Association of Pharmaceutical Scientists

various drugs viz indomethacin-ranitidine hydrochloride(18), in which it has been concluded that both indometha-cin and ranitidine hydrochloride were fully converted intothe amorphous state through molecular interaction after co-milling in contrast to the partially crystalline form of indi-vidually milled drugs. In contrast, the simvastatin-glipizideco-amorphous mixture showed improved physical stabilitywithout evidence of intermolecular interactions as studiedby Lobmann et al. (19). Alleso et al. have illustrated thestabilization of the naproxen-cimetidine amorphous mix-tures dictated by molecular-level interactions rather thanbulk-level phenomena (20). Therefore, there is a hopeand need to rationalize the co-amorphous concept by usinga specific clinical dose ratio of pharmacologically relevantdrug combinations.

In this present work, an attempt has been made toprepare co-amorphous mixture (COAM) of Nateglinide(NT) and Metformin hydrochloride (MT) in their clinicaldose ratio. NT is an oral blood-glucose-lowering agent be-longing to the class meglitinides, a class II drug of theBiopharmaceutics Classification System (BCS), having poorsolubility of about 0.008 mg/ml and high permeability (log P,4.2) (21,22). On the contrary, MT (BCS III) shows solubilityin the range of 300 mg/ml over the entire pH range of 1–6.8(23). Several studies have been reported for improving solu-bility of NT including nanoparticles, solid dispersion, andliquisolid system (22,24,25).

Clinically, combination therapy of oral hypoglycemicagents is reasonable, aiming to minimize side effects withthe additional benefit of reduction in the cost of therapy.The combination of NT and MT is rational because itworks synergistically in patients whose hyperglycemia isnot controlled by monotherapy, and interestingly, its co-administration does not affect the pharmacokinetics of ei-ther drug (26,27).

In existing work, mechanically activated (ball-milling) co-amorphization of anti-diabetic agents NT and MT has beenattempted, aiming at improvement in the dissolution of NT.From the manufacturing viewpoint, ball milling is a greentechnology and is superior to solvent approaches (28). In thisstudy, NT and MT are ball milled in varying ratios in order tocheck the feasibility of co-amorphization. Taking into accountdosage regimen of both drugs, 120 mg of NT and 500 mg ofMT, in same dose ratio, study has been carried out further.The degree of amorphization has been investigated by X-raypowder diffraction (XRPD), differential scanning calorimetry(DSC), Fourier transform infrared spectroscopy (FTIR), andSEM studies. Additionally, physical stability and in vitro dis-solution studies have been performed to ensure improveddissolution of NT.

MATERIALS AND METHODS

Materials

Nateglinide has been provided as a gift sample by CiplaLtd., Goa, India, and Metformin hydrochloride was gifted bySanofi Ltd., Gujrat, India. The chemical structure of bothdrugs used in the present study is shown in Fig. 1. All otherreagents used in the study were of analytical grade.

Methods

Ball Milling

Nateglinide and Metformin hydrochloride were milledtogether to prepare COAM using a ball mill (ScientificEngineering Corporation, India, Model- Secor 141 v.s.) havinga stainless steel cylindrical jar. One gram of NT and MTmixture, at 1:1, 1:3, 1:5, and dose ratio (120 mg: 500 mg),was ball milled using 9 mm stainless steel balls at ambienttemperature with 40 RPM. Trials were taken for optimizationof ball-milling time of COAM till 6 h. The crystalline drugswere also ball milled separately for comparison at the samemilling parameters. Physical mixtures (PM) were prepared bytriturating crystalline NT and MTwith a mortar and pestle for10 min in similar proportions. All the samples were stored in adesiccator until further use.

Saturation Solubility Studies

The solubility of NT, MT, and COAM was determined byadding known excess samples to 5 ml of water and a respectivebuffer (buffer pH 1.2, pH 4.5, pH 6.8, pH 7.5) separately, andsamples were shaken on an orbital shaker for 48 h at 37°C(29). Samples were further centrifuged at 7000 RPM for10 min, and supernatant was filtered through a 0.45-μm mem-brane filter. Drug content was determined by HPLC (Agilentmodel-1220 infinity HPLC, with autosampler, a diode arrayand multiple wavelength detector, and OpenLab software). AHypersil ODS C18 (250×4.6 mm), Thermo Fisher Scientific,USA, column was used for the study. For simultaneous anal-ysis of NT and MT, the HPLC method was developed andvalidated with respect to linearity, accuracy, precision, androbustness at our end. For the HPLC method, the mobilephase consisted of methanol and phosphate buffer pH 3.0(pH adjusted with dilute ortho-phosphoric acid) in the ratioof 75:25, with flow rate 0.8 ml/min, injection volume 20 μl, anddetection wavelength 216 nm being used. The study was per-formed in triplicate, and results were averaged.

X-ray Powder Diffraction (XRPD)

X-ray powder diffraction studies were performed forsamples using an Xpert Pro MPD X-ray Diffractometer(Panalytical, Netherlands) through Xcelerator Detector withdiffracted beam monochromator (k=1.5405 Å). Samples werescanned between 0 and 70° (2θ) using an acceleration voltageand current of 40 kV and 40 mA, respectively. The principlepeaks and intensity data were collected and analyzed using thesoftware WINPLOTR (Panalytical, Netherlands). All samplesof crystalline and ball-milled NT, MT, and COAM were sep-arately studied by XRPD.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry study was carried outon model DSC 6220 (SII Nanotechnology, SEIKO, Japan).Calibration of the DSC instrument was carried out usingindium as reference standard. DSC thermograms were obtain-ed under a nitrogen gas flow of 80 ml/min at a rate of 10°C/minof all fresh ball-milled mixtures and active pharmaceutical

674 Wairkar and Gaud

ingredient (API). Approximately 3 to 5 mg of samples wascrimped in an aluminum pan and heated at a rate of 10°C/minfrom 10 to 350°C. Crystallinity calculation of drugs in theCOAM was performed using the following equation givenby Rawlinson et al. (30):

%Crystallinity ¼ ΔHmCOAMΔHmDrug�W

� 100

Where ΔHmCOAM is the enthalpy of the COAM (mJ/mg) and ΔHmDrug is the enthalpy of drug (mJ/mg) and W isthe weight fraction of drug in COAM.

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra of NT, MT, PM, and ball-milled sampleswere recorded using an infrared spectrophotometer, model-Spectrum Rx1 (Perkin Elmer, USA) to evaluate possible mo-lecular interactions between the NT and MT. Approximately2- to 3-mg samples were ground thoroughly with KBr andmixed uniformly, and the spectra were recorded at a spectralresolution of ±4 cm−1 over a range of 400–4000 cm−1.Triplicate scan of each sample was taken.

Scanning Electron Microscopy

Scanning electron microscope, ZEISS Ultra FE-SEM(Germany), was used to obtain scanning electron micropho-tographs of NT, MT, PM, and COAM. An acceleratingvoltage of 15 kV was used. Before taking a microphoto-graph, the samples were coated with gold under an argonatmosphere using a sputter coater Emitech K-100× (QuorumTechnologies Ltd, UK).

In Vitro Dissolution Studies

In vitro drug release studies were performed for NT andCOAM using USP type I dissolution test apparatus(Electrolab, India) for 1 h with a speed of 100 RPM and900 ml of water and pH 1.2 buffer or phosphate buffer pH 6.8as a dissolution medium (multimedia dissolution study) at37°C. One hundred milligrams of samples of the COAMwere placed in the basket, and 5 ml aliquots were withdrawn

at specific time intervals, and the same amount of volumewas replaced with fresh dissolution medium. The sampleswere analyzed using above described HPLC method. Theexperiments were conducted in triplicate, and analysis of datawas carried out using PCP disso V3 software (BharatiVidyapeeth, Poona College of Pharmacy, Pune, India).

Stability Studies

For stability studies, COAM samples were stored in astability chamber (Thermolab Scientific Equipments Pvt.Ltd., India) at 40°C/75°C condition and ambient tempera-ture for 45 days and were periodically removed at 30 and45 days and evaluated for physical appearance, XRPD, andin vitro dissolution. Further, COAM samples were charac-terized for drug content by HPLC analytical method givenin the BSaturation Solubility Studies^ section.

RESULTS

Ball Milling and Saturation Solubility Studies

The experimentally determined solubility of NT, physicalmixture, and COAM in purified water have been given inTable I. The pH-solubility profile revealed that NT showspH-dependant solubility which gradually increases from acid-ic pH to alkaline pH as shown in Fig. 2. Preliminary studieshave shown linear rise in the solubility of COAM as thequantity of MT increases in the COAM containing 1:1, 1:3,

Fig. 1. Molecular structures of a Nateglinide and b Metformin hydrochloride

Table I. Solubility Study of NT

Description of API/formulation Solubility in water (mg/ml)

Crystalline 0.061Ball milled 0.106NT:MT (1:1 ratio) 0.346NT:MT (1:3 ratio) 0.396NT:MT (1:5 ratio) 0.531NT:MT (Dose ratio) 0.423NT:MT physical mixture (dose ratio) 0.195

API active pharmaceutical ingredient, NT Nateglinide, MT Metfor-min hydrochloride

675Co-Amorphous Combination of Nateglinide-Metformin Hydrochloride

dose ratio, and 1:5. It has been depicted that increase in ball-milling time shows gradual rise in the solubility of NT till240 min. It is evident from solubility studies that MT pos-sesses free solubility in the overall physiological pH range,and there is no statistically significant difference observed inthe COAM form.

X-ray Powder Diffraction (XRPD)

Diffractograms of NT shows distinct peaks of crystal-linity at 13.5°, 16.5°, 19.5°, and 24° and for MT at 17°, 22°,28°, 30°, and 37° (Fig. 3). The XRPD pattern of COAMshowed a reduction in the peak intensity for both drugswithin the first 2 h of ball milling which further decreasedafter 4 h of ball milling. When the ball-milling duration wasextended up to 6 h, the characteristic peaks of NTcompletely disappeared for all API ratios, demonstratingconversion to amorphous form; however, low-intensitypeaks of MT were seen in COAM. Similarly, when thedrugs were separately ball milled for 6 h under same ball-milling conditions, enhanced amorphism was noted in NT,compared to MT. Relatively, the rate of amorphization ofNT was faster than MT in COAM.

Differential Scanning Calorimetry (DSC)

The DSC thermogram of NT and MT showed endother-mic transition peaks corresponding to their melting points at131°C and 235°C, suggesting the presence of polymorph B forNT and polymorph A for MT. In PM, NT and MT peaks wereseen at their melting points as anticipated. However, the en-thalpy of both NT and MT has been reduced from 29.8 to6.8 mJ/mg and 389 to 291 mJ/mg, respectively, in PM.Interestingly, the melting endotherm for NT has almost disap-peared, and the endothermic peak for MT was shifted to 221–225°C from 235°C in all ratios of COAM. This supports totaldisappearance of XRPD peaks of NT, as aforesaid. The %crystallinity data was calculated for NT and MT in COAMsample which shows complete amorphization of NT and 63%crystallinity of MT in COAM. The thermogram details foraforesaid discussion can be seen in Fig. 4.

Fourier Transform Infrared Spectroscopy

FTIR spectra of individual drugs and mixtures thereofhave been given in Fig. 5. The characteristic bands of NTwere observed at the wave numbers of 1209–1384 cm−1

justifying the presence of carboxyl and carboxylate groups.In addition to this peak, C-H stretching between 2867 and3030 cm−1, C=O vibration at 1742 cm−1, and the C–NHbending vibration (amide band II) at 1540 cm−1 were alsoseen in the NT spectrum. Similarly, characteristic bands ofMT were observed including N-H stretching (primary andsecondary) vibrations at wave number 3371 and 3173 cm−1.Furthermore, N-H deformation vibrations at 1623–1577 cm−1 and C-N stretching vibration at 1057–1030 cm−1

were also observed in the MT spectrum. Comparison ofFTIR spectra of crystalline NT, MT, and COAM revealedslight broadening of peaks at the 1750- to 1700-cm−1 regionof COAM as compared to pure drugs.

Scanning Electron Microscopy

Prior to milling, needle-shaped, long, and slender crystalsof different sizes were seen in the NT samples, while irregular-shape crystals were observed in the MT samples, as seen inFig. 6. COAM showed small spheres without any traces ofneedle-shape crystals ensuring the possibility of instantaneousformation of microstructured particles dispersed in the MTmatrix, wherein ordered needle geometry has been lost.Microphotograph of PM as raw material showed mixed irreg-ular and needle structures indicating existence of original formof both API.

In- Vitro Dissolution Studies

Comparison of in vitro dissolution (water media) profilesof crystalline NT, ball-milled NT, physical mixture, andCOAM of dose ratio revealed that crystalline and ball-milledAPI showed very slow drug release rate whereas physicallymixed NT and MT resulted in a further improvement in therelease rate as seen in Fig. 7. However, it hindered the drugrelease over 49.65% at 60 min. COAM of dose ratio exhibitedsuperior dissolution profile in water than the physical mixtureand crystalline/ball-milled NT.

Multimedia drug release profiles of crystalline NT, PM,and COAM are illustrated in Fig. 8. Drug release of COAMwas significantly higher (P<0.05) in pH 1.2 buffer as comparedto crystalline NT or PM. However, there was no major changeobserved in drug release of PM and COAM at higher pHdissolution medium, precisely in phosphate buffer pH 6.8, atthe end of 60 min (Fig. 8). Predictably, Metformin hydrochlo-ride release remained unaffected in multimedia dissolution,and more than 95% drug release was observed of crystallineAPI and in COAM form.

Stability Studies

X-ray powder diffractograms of sample subject to stabil-ity studies have shown no characteristic peaks of NT at 40°C/75°C for 45 days as seen in Fig. 3(H). As partial amorphismwas attained for MT initially, low-intensity peaks of MT havebeen observed in the diffractogram. The in vitro dissolution

Fig. 2. pH solubility profile of NT

676 Wairkar and Gaud

studies also showed satisfactory dissolution profile matchingto the initial samples as shown in Fig. 8. Besides, it was alsonoted that COAM remained as a white to off-white free-flowing powder without changing its physical appearance onstability. Drug content was found in the accepted range of96.4–98.3%w/w for NT and 98.2–99.6%w/w for MT whichwas similar to the initial values for both API.

DISCUSSION

In the present study, the combination of NT and MT hasbeen co-processed in dose ratio by ball milling to form aCOAM. It has been reported that induction of amorphismby mechanical activation can be related to the milling timeand intensity (18,31). Hence, ball-milling time was optimized,and a steady rise in the solubility of NT was observed till240 min. Thereafter, further lengthening the ball-milling du-ration does not show any noteworthy change in the solubilityof COAM.

The pH-dependant solubility of NT at the physiologicalpH range can be attributed to the anionic nature of NT (pKa

value 3.1). NT exists predominantly in un-ionized form at theacidic pH of the stomach which causes very low solubility of adrug, whereas higher physiological pH causes complete ioni-zation of the carboxylic group and improved solubility to alarge extent (32,33). Result suggests that each NT moleculemay be interacting with more than one MT molecule andthereby increases the solubility of NT with the rise in quanti-ties of MT. Thus, increase in the solubility would result inincreased dissolution rate of NT and would resolve the prob-lem of low bioavailability. Being a BCS class III drug, MTshows free solubility in all forms.

Solubility data of COAM is further supported by XRPDstudies which reveal a steady decrease in peak intensity of NTwith time and after 6 h complete amorphization of NT hasbeen attained in COAM. This means a decrease in crystallin-ity of NT leads to improved solubility. On the other hand,partial amorphization of MT was observed which can be con-tributed to its higher quantity in dose ratio of COAM.Surprisingly, NT alone could not be completely transformedinto its amorphous solid form upon ball milling. This can beexplained with Nateglinide structural conformation, stabilized

Fig. 3. X-ray powder diffractogram for a crystalline MT, b ball-milled MT, c crystalline NT,d ball-milled NT, e COAM of dose ratio at 6 h, f COAM of dose ratio at 4 h, g COAM of

dose ratio at 2 h, and h COAM at 40°/75% RH for 45 days

677Co-Amorphous Combination of Nateglinide-Metformin Hydrochloride

through intramolecular hydrogen bonds as reported byBharatam et al. (34). Similar observations have also beenreported by Chieng et al. for indomethacin-ranitidine hydro-chloride in which individual drugs retained partial crystallinityafter co-milling under the same conditions (18).

DSC output had revealed miscibility of NT and MT intoCOAM in addition to the amorphization shown by XRPDstudies. And, a little shift in the melting endotherm withreduction in enthalpy of fusion 221–237 J/g has been notedfor MT, which is an indicator of molecular-level interaction

between NT and MT, which even confirms partial amorphismof MT in COAM. Early amorphization of NT can be attribut-ed to its comparatively small amount of enthalpy, than MT.This observation has also been backed up by % crystallinitydata, which confirms retention of crystallinity of MT inCOAM and no evidence of crystalline NT.

For better understanding of interactions between NT andMT, molecular structures of both have been depicted in Fig. 1.NT has two strong proton acceptors and donors each, whereasMT has four proton acceptors and five donors, enabling

Fig. 4. DSC thermogram of a crystalline NT, b crystalline MT, c physical mixture of NTand MT, d COAM of1:1 ratio, e COAM of 1:3 ratio, and f COAM of dose ratio

Fig. 5. Fourier transform infrared spectra of a crystalline NT, b crystalline MT, c COAM of 1:1 ratio, dCOAM of 1:3 ratio, and e COAM of dose ratio

678 Wairkar and Gaud

hydrogen interactions between both drugs. Thus, hydrogenbonding is responsible for molecular interaction between NTand MT which further influences the arrangement of mole-cules in the COAM (34,35). Generally, band shift and bandbroadening have been observed in the transformation of crys-talline to amorphous compounds (36). Furthermore, specificchanges are indicators of hydrogen bonding between bothdrugs from the vibration regions of the hydrogen-bondedcarboxylic acid moiety (1750- to 1700-cm−1 region), showingprominent broadening of spectra. Thus, IR spectra confirmedphysical interaction between NT and MT, in PM and COAM.

Consequently, SEM microphotographs also supportfindings of XRPD and DSC data and amorphization ofNT. Therefore, it is possible that complete disappearanceof crystalline NT due to physical contact with the hydro-philic MT may be responsible for the enhanced drug solu-bility of the COAM.

Faster drug release rate of COAM in contrast tocrystalline NT cannot be explained merely by particle-size changes during the ball-milling process but can beattributed to an amorphous form of drug. As shown byLöbmann et al., binary co-amorphous drug mixtures

Fig. 6. SEM of a crystalline NT, b crystalline MT c COAM of dose ratio and d physical mixture of MT andNT

Fig. 7. In vitro dissolution profile of NT

679Co-Amorphous Combination of Nateglinide-Metformin Hydrochloride

formed through molecular interactions lead to synchro-nized, specifically pairwise, dissolution (37). Thus, it canbe proposed in effect that the heterodimer of NT-MT isreleased instead of the individual drug. It has been alsohypothesized that the wettability of small lipophilic NTparticles was further improved in the presence of highlysoluble MT (38,39).

Selection of an appropriate dissolution medium shouldbe based on drug characteristics and formulation featureswhich can clearly discriminate the variations in releaseprofile (40,41). The pH-solubility profile suggested thatvery low solubility of NT in the pH 1.2 buffer may leadto the precipitation of NT in the acidic environment of thestomach. Therefore, pH 1.2 buffer has been used as a pH-simulated medium for dissolution of crystalline NT, PM,and COAM as shown in Fig. 8. Moreover, dissolutionparameters should also consider pH at the site of absorp-tion for the drugs showing pH-dependent solubility (42–44).Higher NT release into the pH 1.2 buffer in COAM formis particularly encouraging because dissolution is going tobe a rate-limiting step particularly in the acidic pH of thestomach for immediate release formulations of NT (45,46).However, there was no major difference observed in thedrug release of PM and COAM at a higher pH dissolutionmedium, precisely in phosphate buffer pH 6.8, at the endof 60 min (Fig. 8). This observation was in line with thehigher solubility of NT in alkaline pH. MT has showedalmost complete drug release irrespective of dissolutionmedium. The results were well anticipated and can becredited to its free solubility (47).

Thus, XRPD spectra indicate absence of devitrificationfor NT in COAM and suggest the stabilization of amor-phous NT by MT. Stability samples do not show any differ-ence in in vitro dissolution studies supporting thestabilization of amorphous NT. In addition, physicochemi-cal stability of COAM has been substantiated by the unaf-fected physical appearance and drug content. Hence, asstated above, the increased stability and enhanced dissolu-tion in COAM can be contributed to molecular interactionsbetween NT and MT (48,49).

CONCLUSION

Nateglinide and Metformin hydrochloride have beentransformed into a co-amorphous state by a simple, green,economic ball-milling process. In co-amorphous form, thenon-bonding type of interactions (proton exchange) betweenNT and MT has significantly increased the drug release ratefor Nateglinide in COAM. Hence, the combination therapy ofboth drugs in the form of COAM appears to be an excellentoption for chronic therapy of type 2 diabetes. Thus, the role ofco-amorphism can be further investigated for fixed-dose com-binations of drugs available clinically.

ACKNOWLEDGMENTS

Authors are also thankful to Tata Institute of Fundamen-tal Research (TIFR), India, for assistance on XRPD and SEManalysis.

Conflict of Interest The authors declare that they have no com-peting interests.

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681Co-Amorphous Combination of Nateglinide-Metformin Hydrochloride